ABSTRACT
In this report, we have compared the changes in the production of tRNA(iMet) (initiator tRNA(Met] and tRNA(Asn), which occur during erythroid differentiation in the Friend erythroleukemia cell. The relative steady-state concentration of these two tRNAs (relative to the total tRNA population) was measured by aminoacylation. The results show that while the relative steady-state concentration of tRNA(iMet) changes very little in the cytoplasmic tRNA population, the relative concentration of tRNA(Asn) decreases during the first two days of differentiation and then undergoes an increase. This difference in the behavior of these two tRNAs is also seen when their relative concentrations in newly synthesized tRNA is examined. When tRNA is labeled with tritiated uridine for 24 h in vivo prior to isolation, the hybridization of this labeled tRNA to filter-bound tRNA genes shows that the relative concentration of tRNA(iMet) in newly synthesized tRNA changes very little, while the relative concentration of newly synthesized tRNA(Asn) again decreases through the first 2 days of differentiation, and then undergoes a smaller increase. Thus, the production of these two tRNAs appears to be independently regulated. Independent regulation of synthesis is also observed when examining the production of these two tRNAs in isolated nuclei. During erythroid differentiation, the relative synthesis of tRNA(iMet) (relative to total nuclear RNA synthesis) remains constant, while the relative synthesis of tRNA(Asn) undergoes periodic increases and decreases in value.
Subject(s)
Leukemia, Erythroblastic, Acute/metabolism , RNA, Transfer, Amino Acid-Specific/biosynthesis , RNA, Transfer, Asn/biosynthesis , RNA, Transfer, Met/biosynthesis , Animals , Cell Differentiation , Cell Line , Cell Nucleus/metabolism , Erythroblasts/metabolism , Friend murine leukemia virus , MiceABSTRACT
The production of tRNA(iMet) during Friend cell erythroid differentiation has been studied. In vitro measurements of total nuclear RNA synthesis in nuclei isolated from Friend cells at different stages of differentiation show the total RNA synthesis increases 1.5-fold at day 1 of induction and then decreases through days 2 and 3 to approximately 75% of its rate of synthesis in the nuclei of uninduced cells. The synthesis of RNA polymerase III transcripts undergoes a similar fluctuation through day 2 of induction, but increases again at day 3. The specific synthesis of tRNA(iMet) was measured by hybridization of labelled nuclear RNA to a tRNA(iMet) gene probe. During erythroid differentiation the percentage of nuclear RNA represented by tRNA(iMet) remains constant (0.065%), so that the absolute synthesis of tRNA(iMet) fluctuates during differentiation, in the fluctuations in the synthesis of total nuclear RNA. The relative synthesis of tRNA(iMet) in vivo was studied by labelling cells with 32Pi, isolating the resulting radioactive tRNA--5S RNA population, and hybridizing this population to a tRNA(iMet) gene probe. The ratio of tRNA(iMet)/total tRNA--5S RNA in newly synthesized cytoplasmic RNA remains similar throughout differentiation (averaging 0.0171), implying that the fluctuations observed in the nuclear synthesis of tRNA(iMet) during differentiation probably also occur for the nuclear synthesis of most tRNA and 5S RNA species. Attempts were made to measure the relative steady-state concentration of tRNA(iMet) using both aminoacylation and in vitro end labelling of tRNA followed by hybridization to a tRNA(iMet) gene probe. These two methods gave different results and we discuss the possible pitfalls of using enzymatic methods for quantitating tRNA concentrations in the cell.
Subject(s)
Leukemia, Experimental/pathology , RNA, Transfer, Amino Acyl/biosynthesis , Animals , Cell Differentiation , Cell Line , Kinetics , Leukemia, Erythroblastic, Acute/metabolism , Leukemia, Erythroblastic, Acute/pathology , Leukemia, Experimental/metabolism , Mice , Nucleic Acid Hybridization , RNA, Transfer, Amino Acyl/genetics , Transcription, GeneticABSTRACT
We have used the gene for tRNAMet1 as a hybridization probe to measure the production of tRNAMet1 in the Friend erythroleukemia cell. In this cell, the relative concentration of tRNAMet1 (i.e., the percentage of total steady-state tRNA representing tRNAMet1) is 1.60 +/- 0.18. To study the relative synthesis of tRNAMet1, cells were labeled in vivo with [3H]uridine for periods ranging from 4 to 24 h, and the tRNA was isolated. The fraction of newly-synthesized tRNA representing tRNAMet1 (1.72% +/- 0.11) does not change when different in vivo labeling times are used. This value is similar to the relative concentration of tRNAMet1 in the older steady-state tRNA (1.61% +/- 0.18). The similar relative synthesis values using different labeling times, plus evidence presented that the total tRNA population decays homogeneously (t 1/2 = 110 h) indicate that tRNAMet1 has a cytoplasmic stability similar to the general tRNA population, and that its concentration relative to the tRNA population is established within the nucleus or soon after exiting the nucleus. Measurements of the synthesis of tRNAMet1 in isolated nuclei, relative to the synthesis of total RNA polymerase III transcripts, showed that this relative synthesis (0.291% +/- 0.017) is only 17% of the relative concentration of tRNAMet1 in the cytoplasm, which may reflect the presence of sequences other than tRNA in total nuclear polymerase III transcripts.
